Traumatic Brain Injury (TBI) is the result of external forces impacting the brain. Despite scientific progress, TBI remains a significant cause of impairment and mortality. Recently, laser-induced shockwave (LIS) has emerged as an effective method for TBI simulation. LIS generates shockwaves through pulsed laser-induced plasma formation, allowing for the controlled study of TBI at the cellular level. This study introduces a novel approach to examine cellular morphological changes in response to shear stress, focusing on astrocyte cell type AST-1, by combining LIS with quantitative phase microscopy (QPM). QPM is a label-free technique that allows for real-time cellular dynamics observation through 3D imaging. Integrating LIS and QPM assesses astrocyte responses to shear stress caused by LIS, revealing both immediate and sustained morphological changes. Post-LIS exposure analysis shows significant alterations in astrocyte circularity, volume, surface area, and other features. Statistical tests confirm these observed trends, providing valuable insights into astrocyte responses to mechanical forces. These findings enhance our understanding of how mechanical stimuli affect astrocyte morphology, which may offer the potential for identifying and developing therapeutic strategies in TBI and related neurological disorders.
Biological studies are increasingly using optical forces to study cellular behavior and intracellular interactions. In this talk, we discuss the use of optically generated forces in biomedical treatment and diagnostics with specific reference to traumatic brain injury (TBI). Our biophotonic workstation includes optical tweezers, quantitative phase microscopy, fluorescence imaging, and laser-induced shockwaves to study cellular damage. We discuss our studies on astrocyte damage repair mechanisms as well as how the optical toolbox enables the study of intracellular signaling and cellular dynamics and anatomy.
The use of laser nanosurgery to induce photolysis has proven to be an indispensable tool for in vitro studies of astrocytes. Here, we utilize laser nanosurgery to initiate damage within single astrocytes in an vitro traumatic brain injury model. Changes in cytoplasmic ATP levels were observed throughout the astrocyte network following the targeted lysis of a single cell. In response to the death of a neighboring cell, a transient drop in cytoplasmic ATP levels was observed. This combined method of optical technologies should prove valuable in understanding astrocytes’ role in detection of nervous tissue damage.
There is a need for new methodologies to investigate cell apoptosis and recovery, cell adhesion, and cell-cell interactions in cellular biology and neurobiology. Such systems should be able to induce localized cell injuries and measure damage responses from single cells. In this regard, pulsed lasers can be used to produce Laser- Induced Shockwaves (LIS), which can cause cell detachments and induce cellular membrane injuries, by applying shear force in order of µN . Furthermore, since the resulting shear force can increase membrane permeability, chemicals and markers can then be transferred into cells non-invasively. Continuous-wave lasers can be used as Optical Tweezers (OT), to apply non-contact delicate forces, as low as 0.1f N , and deliver materials into cells, and also move the cells to different locations. In this paper, we introduce a combination of modalities to apply variable forces, from femto to micro newtons, to cells. Our system consists of a 1060nm continuous laser light source for OT and a 1030nm femtosecond pulsed laser for generating LIS. To have a direct measurement of changes in the cellular thickness and membrane dynamics, the cells are imaged under a Quantitative Phase Microscope (QPM). Our microscope is capable of Differential-Interference Microscopy (DIC) and Phase-Contrast microscopy (PhC) and fluorescent microscopy, making it a unique system for studying cell injuries.
Traumatic brain injury (TBI) occurs when an external shock causes injury to the brain. The mechanism of the disease is not completely understood yet. Studies have shown that astrocytes play various roles following brain injury. However, the exact functional role of them after TBI is still a matter of debate. Laser-induced shock waves (LIS) can create a precise controllable mechanical force that is capable of injuring or lysing cells to simulate the brain injury at the cellular level. Here, we propose a system that enables us to induce injuries in CNS cells with LIS and observe the whole process under a Quantitative phase microscope (QPM). Our system is also capable of adding another laser for optically trapping the cells to keep them at a certain distance from the center of the shockwave, as this distance is one of the important factors which determines the level of injury.
In two previous studies we have conducted combined laser subcellular microsurgery and optical trapping
on chromosomes in living cells1, 2. In the latter study we used two separate microscopes, one for the trap
and one for the laser scissors, thus requiring that we move the cell specimen between microscopes and
relocate the irradiated cells. In the former paper we combined the 1064 nm laser trap and the 532 nm laser
scissors into one microscope. However, in neither study did we have multiple traps allowing for more
flexibility in application of the trapping force. In the present paper we describe a combined laser scissors
and tweezers microscope that (1) has two trapping beams (both moveable via rapid scanning mirrors (FSM-
300, Newport Corp.), (2) uses a short pulsed tunable 200 fs 710-990 nm Ti:Sapphire laser for laser
microsurgery, and (3) also has the option to use a 337 nm 4 ns UV laser for subcellular surgery. The two
laser tweezers and either of the laser ablation beams can be used in a cell surgery experiment. The system
is integrated into the robotic-controlled RoboLase system3. Experiments on mitotic chromosomes of rat
kangaroo PTK2 cells are described.
Optical coherence tomography (OCT) is a noninvasive, high-resolution imaging technology capable of delivering real-time, near-histologic images of tissues. Mustard gas is a vesicant-blistering agent that can cause severe and lethal damage to airway and lungs. The ability to detect and assess airway injury in the clinical setting of mustard exposure is currently limited. The purpose of this study is to assess the ability to detect and monitor progression of half-mustard [2-chloroethylethylsulfide (CEES)] airway injuries with OCT techniques. A ventilated rabbit mustard exposure airway injury model is developed. A flexible fiber optic OCT probe is introduced into the distal trachea to image airway epithelium and mucosa in vivo. Progression of airway injury is observed over eight hours with OCT using a prototype time-domain superluminescent diode OCT system. OCT tracheal images from CEES exposed animals are compared to control rabbits for airway mucosal thickening and other changes. OCT detects the early occurrence and progression of dramatic changes in the experimental group after exposure to CEES. Histology and immunofluorescence staining confirms this finding. OCT has the potential to be a high resolution imaging modality capable of detecting, assessing, and monitoring treatment for airway injury following mustard vesicant agent exposures.
In this study, 532-nm picosecond and 800-nm femtosecond lasers are used in combination with fluorescently labeled tubulin to further elucidate microtubule depolymerization and the effect lasers may have on the resulting depolymerization. Depolymerization rates of targeted single microtubules are dependent on location with respect to the nucleus. Microtubules located near the nucleus exhibit a significantly faster depolymerization rate when compared to microtubule depolymerization rates near the periphery of the cell. Microtubules cut with the femtosecond laser depolymerize at a slower rate than unirradiated controls (p=0.002), whereas those cut with the picosecond laser depolymerize at the same rate as unirradiated controls (p=0.704). Our results demonstrate the ability of both the picosecond and femtosecond lasers to cut individual microtubules. The differences between the two ablation results are discussed.
An 800-nm 200-fs laser is used to produce DNA damage in rat kangaroo (PtK1) and human cystic fibrosis pancreatic adenoma carcinoma (CFPAC-1) cells. Immunofluorescence staining for DNA repair factors in irradiated cells displays localization of H2AX, Nbs1, and Rad50 to the site of irradiation 3 to 30 min following laser exposure. It is concluded that the 200-fs near-infrared laser is an excellent source for the production and study of spatially defined regions of DNA damage.
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